Synthesis, characterization and biological evaluation of some novel 17-isoxazoles in the estrone series

Regioselective 1,3-dipolar cycloadditions of different aryl nitrile oxides to mestranol were carried out to furnish novel steroidal 17α-isoxazoles in good to excellent yields. Copper(I) was found to be an efficient catalyst, accelerating the intermolecular ring-closures and leading exclusively to 3,5-disubstituted isoxazoles. The yields of the cycloadducts, however, were influenced by the substituents on the aromatic moiety of the 1,3-dipoles. Moreover, dehydration of the primary products resulted in the corresponding Δ(16,17)exo-heterocyclic derivatives. All the synthesized compounds were subjected to in vitro pharmacological studies of their antiproliferative effects relative to three human malignant cell lines (HeLa, MCF7 and A2780).     

Enzymatic Synthesis of Polyol- and Masked Polyol- Glycosides using β-Glycosidase of Sulfolobus Solfataricus

The use of commercially available mesophilic glycosidases in the enzymatic synthesis of glycosides of different types is a well established method suffering from some drawbacks such as a poor yield. Substrates with three or four hydroxyl groups have been subjected to enzymatic glucosylation using crude homogenate of the thermophilic archaeon Sulfolobus solfataricus containing a β-glycosidase activity able to transfer glucose, galactose and fucose from different donors. The stereochemistry of this reaction was interpreted in terms of interaction with a possible “glucose” active site of the enzyme. In addition masked or protected derivatives of tetritols and some simple unsaturated alcohols were glycosylated yielding glycosides in yields very competitive with those obtained using mesophilic enzymes, examples of further chemical manipulation of these compounds were reported. When using a scarce amount of acceptor, a reasonable amount of products could be obtained by adding different aliquots of donor at time intervals.     

Production of alditols fromd-xylose by yeasts

Production of glycerol, tetritols, pentitols, hexitols and heptitols was tested with 193 strains of yeasts and yeast-like microorganisms belonging to 13 genera. According to the production of alditols, the yeast species were divided into four groups. The largest group consisted of pentitol-producing yeasts. Only few species produced glycerol, tetritol and hexitol. Production of heptitols was found mainly in sporulating yeasts. Translated by Č. Novotny     

Survival of water stress in annual fish embryos: dehydration avoidance and egg envelope amyloid fibers

Diapausing embryos of Austrofundulus limnaeus survive desiccating conditions by reducing evaporative water loss. Over 40% of diapause II embryos survive 113 days of exposure to 75.5% relative humidity. An early loss of water from the perivitelline space occurs during days 1-2, but thereafter, rates of water loss are reduced to near zero. No dehydration of the embryonic tissue is indicated based on microscopic observations and the retention of bulk (freezable) water in embryos as judged by differential scanning calorimetry. Such high resistance to desiccation is unprecedented among aquatic vertebrates. Infrared spectroscopy indicates frequent intermolecular contacts via beta-sheet (14%) in hydrated egg envelopes (chorions). These beta-sheet contacts increase to 36% on dehydration of the egg envelope. Interestingly, the egg envelope is composed of protein fibrils with characteristics of amyloid fibrils usually associated with human disease. These features include a high proportion of intermolecular beta-sheet, positive staining and green birefringence with Congo red, and detection of long, unbranched fibrils with a diameter of 4-6 nm. The high resistance of diapause II embryos to water stress is not correlated with ontogenetic changes in the egg envelope.     

Bile acids of a 3200-year-old Egyptian mummy.

The bile acids of the gall bladder and hepatic tissue of a 3200-year-old Egyptian mummy were isolated by thin-layer chromatography and identified by combined gas-liquid chromatrography and mass spectrometry. Except for complete deconjugation and extensive dehydration, the bile acids were found to correspond in their qualitative and quantitative composition to the gall bladder bile acids of modern man. The secondary bile acids constituted about 50% of the total and were identified as the normal bacterial oxidoreduction products of the primary bile acids and their dehydration products. In addition a series of unsaturated bile acids were identified, which corresponded to the dehydration products of cholic and chenodeoxycholic acids. It is suggested that both bile acid deconjugation and the limited oxidoreduction were probably brought about by the Clostridium organisms identified in the tissue. On the basis of the bile acid composition it is concluded that the ancient man metabolized cholesterol along the same pathways as modern man.     

Mono- and bifunctional binding of cis-diamminedichloroplatinum(II) to dinucleotides

cis-Diamminedichloroplatinum(II) (cis-Pt) was reacted with four homodinucleotides (GpG, ApA, CpC, and UpU) and six heterodinucleotides (GpC, CpG, GpU, UpG, GpA, and ApG) at pH 6, and the reaction products were purified by HPLC. The most important products were characterized by 1H-NMR spectra. In all the heterodinucleotides except the ones containing uridine the main Pt-adduct was an intramolecular cross-link, but monofunctional adducts and intermolecular cross-links were also detected. Intramolecular cross-links were also formed with GpU and UpG but the amounts of them were about the same as the amounts of intermolecular cross-links. In the case of homodinucleotides GpG gave almost entirely intramolecular cross-links, in which cis-Pt was chelated between the N-7 atoms of two guanines. cis-Pt reacted also with ApA forming both monofunctional and bifunctional Pt-adducts. The main adducts were intramolecular cross-links. cis-Pt reacted equally well with all guanosine-containing dinucleotides, while the reaction with ApA was much slower. With CpC and UpU no reaction products were formed.     

Trichiliadregeana胚轴的脱水敏感性与抗坏血酸的抗氧化作用

以顽拗性TrichiliadregeanaSond.种子为材料,研究其胚轴的脱水敏感性与抗坏血酸的抗氧化作用。T.dregeana胚轴的脱水耐性随着脱水进程逐渐下降,50%的胚轴被脱水致死的含水量(W50)大约为0.16gH2O/gDW。在脱水过程中,胚轴的电解质渗漏速率逐渐增加,超氧物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)、过氧化氢酶(CAT)、谷胱苷肽还原酶(GR)和脱氢抗坏血酸还原酶(DHAR)的活性下降,硫代巴比妥酸(TBA)-活性产物的含量增加。2.5~10.0mmol/L抗坏血酸处理能有效地增加胚轴的脱水耐性和SOD、APX、CAT和GR的活性,降低电解质渗漏速率和TBA活性产物的含量。结果表明,T.dregeana胚轴的脱水耐性与抗氧化酶的活性增加和脂质过氧化作用的降低密切相关。     

Preparation and mass spectral behaviour of some 5 beta-cholenoic acids.

Monounsaturated 5 beta-cholanoic acids with double bonds in rings A, B, and C were prepared by POCl3 and ZnCl2 dehydration from natural bile acids with selectively blocked hydroxyl functions. The yields ranged from 15 to 100%. The products were purified by thin-layer and AgNO3 thin-layer chromatography and the structures were confirmed by nuclear magnetic resonance spectroscopy and mass spectrometry. The methyl ester acetates of the unsaturated 5 beta-cholanoic acids possessed chromatographic properties closely similar to those of the corresponding saturated bile acids. Several characteristic fragments were seen in the mass spectra which, in conjunction with the chromatographic properties, permitted an unambiguous distinction between different monounsaturated acids, and between saturated and unsaturated bile acids of the same number and configuration of functional groups. The 20 5 beta-cholenoic acids examined represent all of the simple chemical and enzymatic dehydration products of natural bile acids and can be completely identified by their combined chromatographic and mass spectral properties.     

Thermal stabilization of collagen in skin and decalcified bone

The state of collagen molecules in the fibres of tail tendon, skin and demineralized bone has been investigated in situ using differential scanning calorimetry (DSC). Hydroxyproline analysis and tissue digestion with bacterial collagenase and trypsin were used to confirm that the common cause of all the DSC endotherms was collagen denaturation. This occurred within a narrow temperature range in tendons, but over a wide temperature range in demineralized bone and old skin and demonstrated that in tendon and demineralized bone at least the same type I collagen molecule exists in different thermal states. Hypothesizing that this might be caused by different degrees of confinement within the fibre lattice, experiments were performed to measure the effect of changing the lattice dimensions by extracting the collagen into dilute solution with pepsin, swelling the lattice in acetic acid, and contracting the lattice by dehydration. A theoretical analysis was undertaken to predict the effect of dehydration. Results were consistent with the hypothesis, demonstrating that collagen molecules within the natural fibres of bone and old skin are located at different intermolecular spacings, revealing differences between molecules in the magnitude of either the attractive or repulsive forces controlling their separation. One potential cause of such variation is known differences in covalent cross-linking.     

Possible Involvement of Reactive Oxygen Species Scavenging Enzymes in Desiccation Sensitivity of Antiaris toxicaria Seeds and Axes

The relationships among desiccation sensitivities of Antiaris toxicaria seeds and axes, changes in activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR) and dehydroascorbate reductase, (TBA)-reactive substance were studied. Desiccation tolerance of seeds and axes decreased with dehydration. Desiccation tolerance of axes was higher than that of seeds, and that of epicotyls was higher than radicles. Activities of SOD, CAT and DHAR of seeds increased during the initial phase of dehydration, and then decreased with further dehydration, whereas activities of APX and GR decreased with dehydration. These five enzyme activities of axes, however, increased during the initial phase of dehydration, and then decreased with further dehydration. The rate of superoxide radical production, and the contents of H2O2 and TBA-reactive products of seeds and axes gradually increased with dehydration. These results show that the A. toxicaria seed is a typical recalcitrant seed. Loss of desiccation tolerance in seeds and axes was correlated with activities of seeds and axes.     

Crosslinking of Soybean 7S and 11S Proteins by Transglutaminase

Transglutaminase catalyzes the formation of intermolecular and intramolecular ε-(γ-glutamyl)lysyl crosslinks in proteins. The study here examined the substrate effectiveness of soybean 7S and 11S proteins in the intermolecular-crosslinking reaction catalyzed by guinea pig liver transglutaminase.

Both 7S and 11S proteins could act as the substrate for the transglutaminase reaction. The reaction with 11S protein was faster than that of 7S protein. Analyses of the reaction products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that three main subunit groups of 7S protein and two acidic subunit groups of 11S protein were polymerized through the formation of intermolecular crosslinks by transglutaminase. Interestingly enough, no intermolecular crosslink was formed between the basic subunits of 11S protein. The possible significance of the intermolecular crosslinking catalyzed by transglutaminase is discussed, including the use of this enzyme reaction to improve the properties of food protein.     

Trans interactions between galactosylceramide and cerebroside sulfate across apposed bilayers

The two glycosphingolipids galactosylceramide (GalC) and its sulfated form, cerebroside sulfate (CBS), are present at high concentrations in the multilayered myelin sheath and are involved in carbohydrate-carbohydrate interactions between the lipid headgroups. In order to study the structure of the complex of these two glycolipids by Fourier transform infrared (FTIR) spectroscopy, GalC dispersions were combined with CBS dispersions in the presence and absence of Ca(2+). The FTIR spectra indicated that a strong interaction occurred between these glycolipids even in the absence of Ca(2+). The interaction resulted in dehydration of the sulfate, changes in the intermolecular hydrogen bonding interactions of the sugar and other oxygens, decreased intermolecular hydrogen bonding of the amide C==O of GalC and dehydration of the amide region of one or both of the lipids in the mixture, and disordering of the hydrocarbon chains of both lipids. The spectra also show that Ca(2+) interacts with the sulfate of CBS. Although they do not reveal which other groups of CBS and GalC interact with Ca(2+) or which groups participate in the interaction between the two lipids, they do show that the sulfate is not directly involved in interaction with GalC, since it can still bind to Ca(2+) in the mixture. The interaction between these two lipids could be either a lateral cis interaction in the same bilayer or a trans interaction between apposed bilayers. The type of interaction between the lipids, cis or trans, was investigated using fluorescent and spin-label probes and anti-glycolipid antibodies. The results confirmed a strong interaction between the GalC and the CBS microstructures. They suggested further that this interaction caused the CBS microstructures to be disrupted so that CBS formed a single bilayer around the GalC multilayered microstructures, thus sequestering GalC from the external aqueous phase. Thus the CBS and GalC interacted via a trans interaction across apposed bilayers, which resulted in dehydration of the headgroup and interface region of both lipid bilayers. The strong interaction between these lipids may be involved in stabilization of the myelin sheath.     

Metabolic dysfunction and unabated respiration precede the loss of membrane integrity during dehydration of germinating radicles

This study shows that dehydration induces imbalanced metabolism before loss of membrane integrity in desiccation-sensitive germinated radicles. Using a photoacoustic detection system, responses of CO(2) emission and fermentation to drying were analyzed non-invasively in desiccation-tolerant and -intolerant radicles of cucumber (Cucumis sativa) and pea (Pisum sativum). Survival after drying and a membrane integrity assay showed that desiccation tolerance was present during early imbibition and lost in germinated radicles. However, tolerance could be re-induced in germinated cucumber radicles by incubation in polyethylene glycol before drying. Tolerant and polyethylene glycol (PEG)-induced tolerant radicles exhibited a much-reduced CO(2) production before dehydration compared with desiccation-sensitive radicles. This difference was maintained during dehydration. In desiccation-sensitive tissues, dehydration induced an increase in the emission of acetaldehyde and ethanol that peaked well before the loss of membrane integrity. Acetaldehyde emission from sensitive radicles was significantly reduced when dehydration occurred in 50% O(2) instead of air. Acetaldehyde/ethanol were not detected in dehydrating tolerant radicles of either species or in polyethylene glycol-induced tolerant cucumber radicles. Thus, a balance between down-regulation of metabolism during drying and O(2) availability appears to be associated with desiccation tolerance. Using Fourier transform infrared spectroscopy, acetaldehyde was found to disturb the phase behavior of phospholipid vesicles, suggesting that the products resulting from imbalanced metabolism in seeds may aggravate membrane damage induced by dehydration.     

Mechanism of dehydration inactivation of Lactobacillus plantarum

The mechanism of dehydration inactivation of Lactobacillus plantarum cells after vacuum-drying above saturated salt solutions was studied. The method used is based on the hypothesis that DNase diffuses into cells with damaged cell membranes/walls and hydrolyses the intracellular DNA. Intact, undamaged cells and cells inactivated by either dehydration or heat treatent were incubated in the presence of DNase. The release of DNA hydrolysis products into the incubation medium was measured. It was shown that dehydration inactivation of L. plantarum, but not thermal inactivation, was associated with clear evidence of membrane damage. The residual glucose-fermenting activity of the dehydrated cells related to the release of hydrolysed DNA in the medium, but there was no such relationship with heat-treated cells. Addition of sorbitol to cells before dehydration increased the residual glucose-fermenting activity after drying and this was associated with a reduced rate of DNA hydrolysis. It is concluded that cell wall and/or cell membrane damage is an important mechanism of dehydration inactivation, but that thermal inactivation (up to 60°C) occurs by a different mechanism.Correspondence to: K. van't Riet     

The manifestation of hydrogen bonding in the IR spectra of dl-threitol and erythritol (300–20 K)

The infrared (4000–400 cm⁻¹) and, in part, Raman spectra were recorded for the two isomeric polycrystalline sugar alcohols, dl-threitol and erythritol. Samples were pure substances and isotopically diluted OH/OD compounds. IR spectra were recorded in the 300–20 K range. Assignment of hydrogen bond structure sensitive out-of-plane bending vibrational modes for OH/OD-groups of different H-bond systems is based on isotope exchange and temperature variations. At least seven bands for threitol and two for erythritol correspond to differently H-bonded OH/OD-groups. Relative strengths and quantity of different H-bonds were evaluated. Unlike erythritol, threitol contains over 5% of weak H-bonds. The formation from the melt of a crystalline racemate as a molecular compound of d- and l-forms is suggested. Comparisons with previous neutron scattering results are discussed. In solution, all four OH-groups of both tetritols form H-bonds of equal strength in accord with the basicity of the solvent.     

Changes in the lipid dynamics of liposomal membranes induced by poly(ethylene glycol): free volume alterations revealed by inter- and intramolecular excimer-forming phospholipid analogs.

Influence of osmotic shrinkage, swelling, and dehydration on large unilamellar liposomes (LUVs) of 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC) was investigated using the fluorescent lipid probes 1-palmitoyl-2-[10-(pyren-1-yl)]-decanoyl-sn-glycero-3-phosphocholi ne (PPDPC) and 1,2-bis[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (bisPDPC). Increasing concentrations of poly(ethylene glycol) (PEG, average molecular weight of 6000) producing osmotic gradients delta omega up to 250 mOsm/kg were first added to the outside of LUV labeled with 0.1 mol% of either of the above fluorescent phospholipids. The resulting osmotic shrinkage was accompanied by a progressive reduction in the lateral diffusion of the membrane-incorporated PPDPC, evident as a decrease in the rate of its intermolecular excimer formation. In contrast, under the same conditions the rate of intramolecular excimer formation by bisPDPC increased. Notably, signals opposite to those described above were observed for both of the fluorescent probes upon osmotic swelling of DOPC liposomes with encapsulated PEG. The lateral diffusion of PPDPC became progressively reduced upon membrane dehydration due to increasing concentrations of symmetrically distributed PEG (with equal polymer concentrations inside and outside of the liposomes) when neither shrinkage nor swelling occurs while enhanced excimer formation by bisPDPC was evident. The later results were interpreted in terms of osmotically induced changes in the hydration of lipids. In brief, the removal of water from the phospholipid hydration shell diminishes the effective size of the polar headgroup, which subsequently allows for an enhanced lateral packing of the phospholipid acyl chains. Our findings are readily compatible with membrane free volume Vf changes due to osmotic forces under three different kinds of stress (shrinkage, swelling, and dehydration) applied on the lipid bilayers.     

Identification of glucose-derived cross-linking sites in ribonuclease A

The accumulation of glycation derived cross-links has been widely implicated in extracellular matrix damage in aging and diabetes, yet little information is available on the cross-linking sites in proteins and the intra- versus intermolecular character of cross-linking. Recently, glucosepane, a 7-membered heterocycle formed between lysine and arginine residues, has been found to be the single major cross-link known so far to accumulate during aging. As an approach toward identification of glucose derived cross-linking sites, we have preglycated ribonuclease A first for for 14 days with 500 mM glucose, followed by a 4-week incubation in absence of glucose. MALDI-TOF analysis of tryptic digests revealed the presence of Amadori products (Delta m/ z = 162) at K1, K7, K37 and K41, in accordance with previous studies. In addition, K66, K98 and K104 were also modified by Amadori products. Intramolecular glucosepane cross-links were observed at K41-R39 and K98-R85. Surprisingly, the only intermolecular cross-link observed was the 3-deoxyglucosone-derived DODIC at K1-R39. The identity of cross-linked peptides was confirmed by sequencing with tandem mass spectrometry. Recombinant ribonuclease A mutants R39A, R85A, and K91A were produced, purified, and glycated to further confirm the importance of these sites on protein cross-linking. These data provide the first documentation that both intramolecular and intermolecular cross-links form in glucose-incubated proteins.     

Monolayer autoxidation of arachidonic acid to epoxyeicosatrienoic acids as a model of their potential formation in cell membranes

In light of the importance of epoxyeicosatrienoic acids (EETs) in mammalian pathophysiology, a nonenzymatic route that might form these monoepoxides in cells is of significant interest. In the late 1970s, a simple system of arranging linoleic acid molecules on a monolayer on silica was devised and shown to yield monoepoxides as the main autoxidation products. Here, we investigated this system with arachidonic acid and characterized the primary products. By the early stages of autoxidation (～10% conversion of arachidonic acid), the major products detected by LC-MS and HPLC-UV were the 14,15-, 11,12-, and 8,9-EETs, with the 5,6-EET mainly represented as the 5-δ-lactone-6-hydroxyeicosatrienoate as established by ¹H-NMR. The EETs were mainly the cis epoxides as expected, with minor trans configuration EETs among the products. ¹H-NMR analysis in four deuterated solvents helped clarify the epoxide configurations. EET formation in monolayers involves intermolecular reaction with a fatty acid peroxyl radical, producing the EET and leaving an incipient and more reactive alkoxyl radical, which in turn gives rise to epoxy-hydro(pero)xides and other polar products. The monolayer alignment of fatty acid molecules resembles the arrangements of fatty acids in cell membranes and, under conditions of lipid peroxidation, this intermolecular mechanism might contribute to EET formation in biological membranes.